Helicobacter pylori is a Gram-negative spiral bacterium which infects the human stomach. It is believed that over 50% of the world's population harbour the bacterium.
Clinical isolates of H. pylori can be characterised by the expression of a vacuolating cytotoxin (VacA), which induces vacuole formation in epithelial cells, and an immunodominant cytotoxin-associated antigen (CagA). Type I strains, which predominate in patients with ulcers or cancer, express both of these proteins, whereas type II strains express neither.
Various antigenic proteins have been described for H. pylori, (e.g. references 1&2) including its urease, VacA, flagella proteins, and adhesins. A protein known as NAP, (neutrophil activating protein (3,4)), which is found in both type I and II strains, appears to be protective when tested in the H. pylori mouse model (5).
NAP is a homodecamer of 15 kDa subunits (6), and it has been proposed that the multimeric complex has a ring-shaped structure which spontaneously forms hexagonal paracrystalline structures. The assembled protein appears to interact with glycosphingolipid receptors of human neutrophils (7).
Based on homology with bacterioferritins, it has been suggested that NAP may act as an iron buffer (3). However, the presence of neither iron nor heme has been detected to date. The protein has also been reported to be a Na+/H+ antiporter (8).
As its name suggests, NAP promotes activation and adhesion of neutrophils to endothelial cells. Whilst it is has been suggested that this function is unlikely to be related to its intracellular function (3), the proadhesive activity can be neutralised by antiserum (6). Since neutrophil activation and adhesion to endothelial cells constitute inflammation mechanisms, and since H. pylori is responsible for stomach inflammation, it seems likely that NAP represents the factor, or a factor, of H. pylori responsible for inflammation, probably at an early stage of gastric ulcer disease when an abundant accumulation of neutrophils in the superficial gastric mucosa is observed.
A protocol for the purification of the NAP decamer from H. pylori has been described (6), involving agarose chromatography, molecular sieving and ion-exchange chromatography. This gave a yield of 72%. Recombinant NAP production in E. coli has also been reported (7). The gene was cloned into plasmid pTrxFus to produce a thioredoxin fusion protein. Protein was then purified in the same way as the native protein. The N-terminal thioredoxin was reported not to affect the biological activity of NAP.
There remains, however, a need for pure NAP without the presence of cloning artefacts, fusion domains or the like. It is therefore an object of the invention to provide a process for the purification of native NAP. It is a further object that this process should be straightforward, easily scalable and economically feasible. It is a further object that the process should provide a highly pure protein.
We have now found that NAP has a surprisingly high aqueous solubility, remaining soluble even at 80% ammonium sulphate saturation.